The stacking of chloroplast thylakoids: Evidence for segregation of charged groups into nonstacked regions

Small particles derived from the digitonin treatment of chloroplast thylakoid membranes in either the stacked (grana-containing) or unstacked condition, as determined by cation concentration, have been used to study the aggregation of thylakoid membranes. At pH values above 5, the small particles from stacked chloroplasts do not aggregate in the presence of Mg²⁺ or other screening cations at concentrations sufficient to cause the restacking of thylakoids from low-salt chloroplasts. However, the small particles from stacked chloroplasts are aggregated either by lowering the pH to 4.6 or adding the binding cation La³⁺. In contrast, the small particles obtained on digitonin treatment of unstacked chloroplasts were aggregated by cations at neutral pH. Large particles (mainly grana) derived from digitonin treatment of stacked chloroplasts could not be unstacked by transfer to media of low cation concentration. It is concluded that the nonappressed regions of the chloroplast thylakoid membranes under stacking conditions carry higher than average negative surface charge densities under physiological pH conditions. Transfer of chloroplasts to media of low cation concentration causes a time-dependent lateral redistribution of charge between the appressed and nonappressed regions, but this redistribution is prevented by prior digitonin treatment of stacked chloroplasts.     

Effects of cations on the adhesion between membrane vesicles obtained by digitonin fractionation of spinach chloroplasts

The heavy fraction obtained by digitonin treatment of stacked spinach chloroplasts, suspended in media with different ionic composition, was examined by electron microscopy. In the presence of 5 mM MgCl₂ the thylakoid fragments adhere to one another in a ‘stacked configuration,’ while, in the presence of 10 mM NaCl, mainly only single ‘unstacked’ vesicles are present, which, upon addition of 5 mM MgCl₂, completely revert to the stacked configuration. As previously reported (Chow, W.S. and Barber, J. (1980) Biochim. Biophys. Acta 593, 149–157), no difference in fractionation of chlorophyll between light and heavy fractions was seen after a second digitonin treatment of this fraction suspended in media containing different cation concentrations. From these results it was concluded: (1) that for the unstacking process the movement of proteins or complexes from the stromal to the granal lamellae is not required. Upon lowering the screening by cations of the surface negative charges, the membranes separate from one another; (2) that, under these conditions, as in others (Jennings, R.C., Gerola, P.D., Garlaschi, F.M. and Forti, G. (1980) FEBS Lett. 115, 39–42), digitonin fractionation is not a tool to investigate the degree of membrane stacking.     

The effects of cation-induced and pH-induced membrane stacking on chlorophyll fluorescence decay kinetics

We have compared the effects of thylakoid membrane appression by electrostatic screening and by charge neutralization on the room-temperature chlorophyll fluorescence decay kinetics of broken spinach chloroplasts. Monovalent and divalent metal cations induce both a structural differentiation of thylakoid membranes and a lateral segregation of pigment-protein complexes. These phenomena have distinct effects on the F0- and Fmax-level chlorophyll fluorescence decay kinetics at different levels of added cation. We further find specific cation effects, particularly on a 1-2 ns decay component at the Fmax fluorescence level, that are proposed to be related to the effectiveness of electrostatic screening as determined by the hydrated metal ionic radius. Distinct pH-induced effects on chlorophyll fluorescence decay kinetics are associated with the alternative mechanism of electrostatic neutralization to induce membrane stacking. These observations are used to construct a model of chlorophyll fluorescence emission that accounts for the variable kinetics and multiexponential character of the fluorescence decay upon membrane appression.     

Effect of cations on the reconstitution of heavy subchloroplast fractions (grana) in disorganized low-salt agranal chloroplasts

The disorganization of grana in spinach chloroplasts and their reconstitution has been studied by varying their ionic environment. Dissociation in low-salt media and reconstitution by added cations (monovalent or divalent) was correlated with the formation in high yield of light or heavy subchloroplast membrane fractions, respectively, produced after digitonin treatment of chloroplasts. The formation of heavy subchloroplast fractions was dependent on cation concentration and reached a plateau at 0.1 m monovalent cation or 0.002 m divalent cation. The cation reconstituted fractions recovered the composition and activities of the respective fractions obtained from control chloroplasts. Cation addition to light subchloroplast fractions isolated from low-salt agranal chloroplasts after digitonin disruption also produced heavy fractions. Divalent cations were more effective than monovalent. The heavy fractions produced were enriched in Chlorophyll b and photosystem II activity while the light fractions were enriched in Chlorophyll a and photosystem I activity. The mechanism by which cations induce formation of heavy subchloroplast fractions is not osmotic. Upon reconstitution, stacking of thylakoids seems to occur at specific membrane binding sites.     

Correlation between cation-induced formation of heavy subchloroplast fractions and cation-induced increase in chlorophyll a fluorescence yield in Tricine-washed chloroplasts

A good correlation exists between the extent of thylakoid aggregation (grana reconstitution) and the increase in the chlorophyll a fluorescence yield (F_DCMU; DCMU = 3-(3′,4′-dichlorophenyl)-1, 1-dimethyl urea) caused by the addition of monovalent or divalent cations to low-salt disorganized (agranal) chloroplasts. The extent of grana stacking was monitored by the yield of heavy subchloroplast fractions after digitonin disruption of chloroplasts. A good correlation of the cation effect on both parameters was also found in light subchloroplast fractions (10,000g supernatants) obtained from sonicated “low-salt” Tricine-suspended pea chloroplasts. Addition of cations to the agranal protochloroplasts of etiolated pea or bean leaves exposed to periodic light-dark cycles, suspended in low-salt Tricine buffer, does not affect formation of heavy subchloroplast fractions, nor does it affect their chlorophyll a fluorescence yield level (F_DCMU). The cation effect on the increase of the chlorophyll a fluorescence yield level seems to be due to the cation-induced thylakoid structural changes leading to grana stacking.     

The interaction of an amphipathic fluorescence probe, 2-p-toluidinonaphthalene-6-sulphonate, with isolated chloroplasts.

The amphipathic fluorescence probe, 2-p-toluidinonaphthalene-6-sulphonate has been used to investigate the surface electrical properties of chloroplast thylakoid membranes. The fluorescence yield of 2-p-toluidinonaphthalene-6-sulphonate in aqueous solution increases on addition of hypotonically shocked chloroplast, and the emission maximum shifts towards the blue to 440 nm, although the emission spectrum is somewhat distorted by chloroplast pigment absorption. The intensity of 2-p-toluidinonaphthalene-6-sulphonate fluorescence is further increased on adding salts to the membrane suspension, and changes of greater than 100% are routinely observed. Similar observations have also been made with soya bean phospholipid (azolectin) liposomes. The magnitude of the fluorescence increase is dependent on membrane concentration, being more pronounced at high surface area/suspending volume ratios. The effect of salt addition appears to be that of shielding the fixed negative charges on the membrane surface, thus increasing the fraction of 2-p-toluidinonaphthalene-6-sulphonate molecules at the surface, where the 2-p-toluidinonaphthalene-6-sulphonate has a higher fluorescence yield than in free aqueous solution. This concept is supported by the fact that the effectiveness of salts in increasing 2-p-toluidinonaphthalene-6-sulphonate fluorescence is as predicted by classical electrical double layer theory: governed mainly by the charge carried by the cation with an order of effectiveness C3+ greater than C2+ greater than C+, and not by the chemical nature of the cation or by the nature of its co-ion. It has been argued that the chlorophyll fluorescence yield, controlled by the cation composition of the suspending medium follows the total diffusible positive charge density at the thylakoid membrane surface (Barber, J., Mills, J. and Love, A. (1977) Febs. Lett. 74, 174--181). Although the cation induced 2-p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yield changes show similar characteristics, there are also distinct differences between the two phenomena particularly when cations are added to chloroplasts initially suspended in a virtually cation-free medium. Therefore it is concluded that although both 2-p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yields are governed by the electrical properties of the thylakoid membrane surface, the mechanism controlling their cation sensitivity is not the same.     

A study of factors which regulate the membrane appression of lettuce thylakoids in relation to irradiance

Factors that may influence the extent of thylakoid membrane appression have been examined using lettuce (Lactuca sativa cv. Celtuce) grown under different irradiances. Electron microscopy and salt-induced chlorophyll fluorescence suggest that the percentage of membrane appression is increased in plants grown in low light (20 Wm^–2) compared with those grown in high light (150 Wm^–2). In high light plants surface charge, as measured by 9-aminoacridine, was found to be twice that measured in low light plants. There was a similar difference in ATPase activity of CF₁ and in light saturated photophosphorylation. The chlorophyll content of LHC-2 as a proportion of the total chlorophyll was greatest in thylakoids of low light plants. Measurement of non-cyclic photophosphorylation rates suggested that membrane appression has a stimulatory role in the photophosphorylation process. The importance of these inter-related factors for the mechanism of thylakoid appression is discussed.Abbreviations PS photosystem - chl chlorophyll - LHC-2 light harvesting chlorophyll-protein complex serving PS 2 - CF₁ coupling factor 1 - NADP nicotinamide-adenine dinucleotide phosphate     

Fluorescence changes in isolated broken chloroplasts and the involvement of the electrical double layer

We studied the effects of a variety of cations on chlorophyll fluorescence yield of broken chloroplasts prepared under carefully controlled ionic conditions. In the absence of light-induced electron transport and associated proton pumping, two types of cation-induced chlorophyll fluorescence changes could be distinguished in broken chloroplasts. These are termed "reversible" and "irreversible" fluorescence yield changes. Reversible fluorescence yield changes are characterized by antagonistic effects of monovalent and divalent cations and are prevented by the presence of 5 mM Mg2+ in the suspending media. Reversible-type fluorescence yield changes show little or no dependence on the structure, lipid solubility, or coordination number of the cation, but depend strictly on the net positive charge carried by the ion. It is proposed that these fluorescence changes are brought about through the interaction of monovalent or divalent cations with an electrical double layer at the interface of the outer surface of the thylakoid membrane and the surrounding aqueous solution. The results are interpreted in terms of the Gouy-Chapman theory of the diffuse double layer, indicating that the thylakoid outer surface bears an excess fixed negative charge density of about 2.5 muC/cm2, or approximately 1 negative charge per 640 A2 of membrane surface. Chlorophyll fluorescence quenching in isolated broken chloroplasts suspended in media containing 5 mM MgCl2 is also observed on addition of certain polyvalent cations to the medium. This type of cation-induced fluorescence change appears to be largely irreversible and may occur through specific binding of the cation to the thylakoid as a result of the high electrostatic attraction exerted by the negatively charged membrane surface.     

Investigations of the role of the main light-harvesting chlorophyll- protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex

The functions of the light-harvesting complex of photosystem II (LHC- II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication- freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems.     

Surface potential and reaction of the membrane-bound electron transfer components. II. Integrity of the chloroplast membrane and reaction of P-700

Electrostatic characteristics of the membrane surface in the vicinity of P-700 were estimated by analyzing the salt and detergent effects on its reaction rate with ionic reagents using the Gouy-Chapman diffuse double layer theory in various preparations of chloroplasts.

Upon disruption of thylakoid membranes by sonic treatment or by treatment with digitonin, the reaction rate markedly increased, while the estimated surface charge density became smaller.

It was concluded that the membrane surface which determines the reaction rate between P-700 and the ionic reagents changed as the disruption of thylakoid structure. The outer thylakoid surface had more negative charges than the inner one.

Changes in the electrical potential profile across the thylakoid membrane during the illumination were also discussed from these results.     

Surface potential and reaction of the membrane-bound electron transfer components. II. Integrity of the chloroplast membrane and reaction of P-700

Electrostatic characteristics of the membrane in the vicinity of P-700 were estimated by analyzing the salt and detergent effects on its reaction rate with ionic reagents using the Gouy-Chapman diffuse double layer theory in various preparations of chloroplasts. Upon disruption of thylakoid membranes by sonic treatment or by treatment with digitonin, the reaction rate markedly increased, while the estimated surface charge density became smaller. It was concluded that the membrane surface which determines the reaction rate between P-700 and the ionic reagents changed as the disruption of thylakoid structure. The outer thylakoid surface had more negative charges than the inner one. Changes in the electrical potential profile across the thylakoid membrane during the illumination were also discussed from these results.     

Development of the cation-induced stacking capacity during the biogenesis of higher plant thylakoids

We studied the capacity of the thylakoid membrane to form grana stacks in the presence of cations, monovalent or divalent, added to N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine “low-salt” disorganized plastids during their greening. Grana stacking was monitored by the yield of heavy subchloroplast fractions separated by differential centrifugation after digitonin disruption of plastids (J. H. Argyroudi-Akoyunoglou, 1976, Arch. Biochem. Biophys., 176, 267–274). Primary thylakoids of the agranal protochloroplasts formed in periodic light do not show the cation-induced stacking capacity of the mature green chloroplast thylakoids. Similarly, the cation effect saturates at lower cation concentrations in mature chloroplasts than in plastids of the early stages of greening. The capacity for cation-induced stacking and for saturation of the effect at low cation concentrations appears gradually after exposure to continuous light and parallel to the appearance of chlorophyll b and the polypeptides of the 25,000–30,000 molecular weight range of lipid-free thylakoids, probably derived from the chlorophyll b-rich chlorophyll protein Complex II. The thylakoid peripheral stroma proteins ribulosediphosphate carboxylase and the coupling factor protein are not involved in the cation-induced stacking, since their removal (H. Strotmann, H. Hesse, and K. Edelmann, 1973, Biochim. Biophys. Acta, 314, 202–210) does not affect the thylakoid aggregation.     

3-D modelling of chloroplast structure under (Mg) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll-protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.     

A new type of subchloroplast fragments isolated from pea chloroplasts in the presence of digitonin

Heavy fragments were isolated from pea chloroplasts using digitonin treatment and differential centrifugation. The particles were characterized by a significantly lowered chlorophyll a/b ratio, contents of photosystem I (PS I) proteins and ATPase, as well as of amount of P700. The content of photosystem II (PS II) proteins decreased insignificantly, whereas that of proteins of the light-harvesting complex II did not change. The absorption and low-temperature fluorescence spectra were indicative of a decreased content of PS I. Electron microscopy of ultrathin sections of heavy fragment preparations identified them as grana with reduced content of thylakoids. The diameter of these particles was practically the same as within chloroplasts. Comparison of various characteristics of the fragments and chloroplasts from which the fragments were isolated allowed us to define a high degree of preservation of marginal regions in thylakoids present in the heavy fragment particles. Analysis of the results shows that the procedure of fragmentation produces grana with high extent of thylakoid integrity. The phenomenon of reduction of the thylakoid content in grana, occurring as our heavy fragments, is considered in the frame of our previous hypothesis concerning the peculiarities of grana organization in the transversal direction.     

The interaction of an amphipathic fluorescence probe, 2-p-toluidinonaphthalene-6-sulphonate,with isolated chloroplasts

The amphipathic fluorescence probe, 2-p-toluidinonaphthalene-6-sulphonate has been used to investigate the surface electrical properties of chloroplast thylakoid membranes. The fluorescence yield of 2-p-toluidinonaphthalene-6-sulphonate in aqueous solution increases on addition of hypotonically shocked chloroplast, and the emission maximum shifts towards the blue to 440 nm, although the emission spectrum is somewhat distorted by chloroplast pigment absorption.The intensity of 2-p-toluidinonaphthalene-6-sulphonate fluorescence is further increased on adding salts to the membrane suspension, and changes of >100% are routinely observed. Similar observations have also been made with soya bean phospholipid (azolectin) liposomes. The magnitude of the fluorescence increase is dependent on membrane concentration, being more pronounced at high surface area/suspending volume ratios. The effect of salt addition appears to be that of shielding the fixed negative charges on the membrane surface, thus increasing the fraction of 2-p-toluidinonaphthalene-6-sulphonate molecules at the surface, where the 2-p-toluidinonaphthalene-6-sulphonate has a higher fluorescence yield than in free aqueous solution. This concept is supported by the fact that the effectiveness of salts in increasing 2-p-toluidinonaphthalene-6-sulphonate fluorescence is as predicted by classical electrical double layer theory: governed mainly by the charge carried by the cation with an order of effectiveness C³⁺ > C²⁺ > C⁺, and not by the chemical nature of the cation or by the nature of its co-ion.It has been argued that the chlorophyll fluorescence yield, controlled by the cation composition of the suspending medium follows the total diffusible positive charge density at the thylakoid membrane surface (Barber, J., Mills, J. and Love, A. (1977) Febs. Lett. 74, 174–181). Although the cation induced 2-p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yield changes show similar characteristics, there are also distinct differences between the two phenomena particularly when cations are added to chloroplasts initially suspended in a virtually cation-free medium. Therefore it is concluded that although both 2-p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yields are governed by the electrical properties of the thylakoid membrane surface, the mechanism controlling their cation sensitivity is not the same.     

Simulation of grana stacking in a model membrane system. Mediation by a purified light-harvesting pigment-protein complex from chloroplasts

An isolated light-harvesting pigment-protein complex contains polypeptides which bind chlorophyll a and b. The individual complexes can be purified from detergent-solubilized membranes. The isolated light-harvesting complex, when dialyzed to remove detergents, was examined by freeze-fracture electron microscopy. The material consisted of planar sheets of 80-Å subunits which interacted via an edge-to-edge contact. Addition of cations caused the planar light-harvesting complex sheets to become tightly appressed in multilamellar stacks, with distinct subunits still visible within each lamellar sheet. A transition of particle organization from random to crystalline occurred in parallel with the cation-induced lamellar association. Treatment of the dialyzed light-harvesting complex subunits with low levels of the proteolytic enzyme trypsin removed a 2000 molecular weight segment of the major polypeptide of the light-harvesting complex and blocked all subsequent cation-induced changes in structural organization of the isolated light-harvesting complex lamellar sheets.To gain further evidence for mechanisms of cation effects upon the organization of the light-harvesting complex in native membranes, the light-harvesting complex was incorporated into uncharged (phosphatidylcholine) lipid vesicles. The protein complexes spanned the lipid bilayer and were arranged in either a random pattern or in hexagonal crystalline lattices. Addition of either monovalent or divalent cations to ‘low-salt’ (20 mM monovalent cation) vesicles containing light-harvesting complex caused extensive regions of membrane appression to appear. It is concluded that this cation-induced membrane appression is mediated by surface-exposed segments of the light-harvesting complex since (a) phosphatidylcholine vesicles themselves did not undergo cation-induced aggregation, and (b) mild trypsin digestion of the surface-exposed regions of the light-harvesting complex blocked cation-induced lamellar appression. The particles in the appressed vesicle membranes tended to form long, linear arrays of particles, with occasional mixed quasi-crystalline arrays with an angular displacement near 72°. Surface-mediated interactions among light-harvesting complex subunits of different membranes are, therefore, related to changes in structural organization and interaction of the particles within the lipid phase of the membrane.Numerous previous studies have implicated the involvement of the light-harvesting complex in mediating grana stocking in intact chloroplast membranes. The data presented herein provide a simulation of the membrane appression phenomena using a single class of chloroplast-derived membrane subunits. The data demonstrate that specific surface-localized regions of the light-harvesting complex are involved in membrane-membrane interactions.     

Evidence for the role of surface-exposed segments of the light-harvesting complex in cation-mediated control of chloroplast structure and function.

Chloroplast membranes contain a light-harvesting pigment-protein complex (LHC) which binds chlorophylls a and b. A mild trypsin digestion of intact thylakoid membranes has been utilized to specifically alter the apparent molecular weights of polypeptides of this complex. The modified membrane preparations were analyzed for altered functional and structural properties. Cation-induced changes in room temperature fluorescence intensity and low temperature chlorophyll fluorescence emission spectra, and cation regulation of the quantum yield of photosystem I and II partial reactions at limiting light were lost following the trypsin-induced alteration of the LHC. Electron microscopy revealed that cations can neither maintain nor promote grana stacking in membranes which have been subjected to mild trypsin treatment. Freeze-fracture analysis of these membranes showed no significant differences in particle density or average particle size of membrane subunits on the EF fracture face; structural features of the modified lamellae were comparable to membranes which had been unstacked in a “low salt” buffer. Digitonin digestion of trypsin-treated membranes in the presence of cations followed by differential centrifugation resulted in a subchloroplast fractionation pattern similar to that observed when control chloroplasts were detergent treated in cation-free medium. We conclude that: (a) the initial action of trypsin at the thylakoid membrane surface of pea chloroplasts was the specific alteration of the LHC polypeptides, (b) the segment of the LHC polypeptides which was altered by trypsin is necessary for cation-mediated grana stacking and cation regulation of membrane subunit distribution, and (c) cation regulation of excitation energy distribution between photosystem I and II involves the participation of polypeptide segments of the LHC which are exposed at the membrane surface.     

Electrostatic control of chloroplast coupling factor binding to thylakoid membranes as indicated by cation effects on electron transport and reconstitution of photophosphorylation

1. Increase in electron transport rate and the decay rate of the 518 nm absorption change, induced by EDTA treatment, is prevented by cations. The order of effectiveness is C³⁺ > C²⁺ > C⁺.2. In this respect methyl viologen is an effective divalent cation in addition to its action as an electron acceptor.3. Complete cation irreversible EDTA-induced uncoupling occurs in the dark in 2 min. Light greatly stimulates the rate of uncoupling by EDTA. It is concluded that the uncoupling is due to release of coupling factor I from the thylakoid membrane.4. Binding of purified coupling factor I to coupling factor I-depleted thylakoids can be achieved with any cation. The order of effectiveness is C³⁺ > C²⁺ > C⁺, reconstituted thylakoids are active in photophosphorylation regardless of the cation used for coupling factor I binding.5. The marked difference in the concentration requirements for cation effects on 9-aminoacridine fluorescence yield and for prevention of uncoupling by EDTA indicate that coupling factor I and its binding site have a lower surface charge density than the net surface charge density of the thylakoid membrane.6. It is concluded that coupling factor I binding only occurs when negative charges on coupling factor I and its binding site are electrostatically screened by cations.7. Previously reported examples of uncoupling by low ionic conditions are discussed in relation to the basic concepts of diffuse electrical layer theory.     

Activation of latent phenolase from spinach chloroplasts by ageing and by frost

Activation of latent phenolase by freezing and thawing occurs in both thylakoid sediments and membrane washings from spinach chloroplasts, while ageing and digitonin treatment activates membrane-bound enzyme only. Disc clectrophoresis reveals that frost converts a soluble, latent phenolase to an active form after its release from the thylakoid membrane. Ageing of membranes containing latent phenolase results in direct liberation of other active forms. There are further active, soluble forms, which are exclusively found in the chloroplast stroma fraction.     

Cation-mediated regulation of excitation energy distribution in chloroplasts lacking organized Photosystem II complexes

Despite the total loss of Photosystem II activity, thylakoids isolated from the green nuclear maize mutant hcf1-3 contain normal amounts of the light-harvesting chlorophyll $\text{a}\text{b}$ pigment-protein complex (LHC). We interpret the spectroscopic and ultrastructural characteristics of these thylakoids to indicate that the LHC present in these membranes is not associated with Photosystem II reaction centers and thus exists in a ‘free’ state within the thylakoid membrane. In contrast, the LHC found in wild-type maize thylakoids shows the usual functional association with Photosystem II reaction centers. Several lines of evidence suggest that the free LHC found in thylakoids isolated from hcf1-3 is able to mediate cation-dependent changes in both thylakoid appression and energy distribution between the photosystems: (1) Thylakoids isolated from hcf1-3 and wild-type seedlings exhibit a similar Mg²⁺-dependent increase in the short/long wavelength fluorescence emission peak ratio at 77 K. This Mg²⁺ effect is lost following incubation of thylakoids isolated from either source with low concentrations of trypsin. Such treatment results in the partial proteolysis of the LHC in both membrane types. (2) Thylakoids isolated from both hcf1-3 and wild-type seedlings show a similar Mg²⁺ dependence for the enhancement of the maximal yield of room temperature fluorescence and light scattering; both Mg²⁺ effects are abolished by brief incubation of the thylakoids with low concentrations of trypsin (3) Mg²⁺ acts to reduce the relative quantum efficiency of Photosystem I-dependent electron transport at limiting 650 nm light in thylakoids isolated from hcf1-3. (4) The pattern of digitonin fractionation of thylakoid membranes, which is dependent upon structural membrane interactions and upon LHC in the thylakoids, is similar in thylakoids isolated from both hcf1-3 and wild-type seedlings. We conclude that the surface-exposed segment of the LHC, but not the LHC-Photosystem II core association, is necessary for the cation-dependent changes in both thylakoid appression and energy distribution between the two photosystems, and that the LHC itself is able to transfer excitation energy directly to Photosystem I in a Mg²⁺-dependent fashion in the absence of Photosystem II reaction centers. The latter phenomenon is equivalent to a cation-induced change in the absorptive cross-section of Photosystem I.